Endothelial-Specific Reduction in Arf6 Impairs Insulin-Stimulated Vasodilation and Skeletal Muscle Blood Flow Resulting in Systemic Insulin Resistance in Mice.

BACKGROUND: Much of what we know about insulin resistance is based on studies from metabolically active tissues such as the liver, adipose tissue, and skeletal muscle. Emerging evidence suggests that the vascular endothelium plays a crucial role in systemic insulin resistance; however, the underlying mechanisms remain incompletely understood. Arf6 (ADP ribosylation factor 6) is a small GTPase that plays a critical role in endothelial cell function. Here, we tested the hypothesis that the deletion of endothelial Arf6 will result in systemic insulin resistance. METHODS: We used mouse models of constitutive endothelial cell-specific Arf6 deletion (Arf6f/- Tie2Cre+) and tamoxifen-inducible Arf6 knockout (Arf6f/f Cdh5CreER+). Endothelium-dependent vasodilation was assessed using pressure myography. Metabolic function was assessed using a battery of metabolic assessments including glucose and insulin tolerance tests and hyperinsulinemic-euglycemic clamps. We used a fluorescence microsphere-based technique to measure tissue blood flow. Skeletal muscle capillary density was assessed using intravital microscopy. RESULTS: Endothelial Arf6 deletion impaired insulin-stimulated vasodilation in white adipose tissue and skeletal muscle feed arteries. The impairment in vasodilation was primarily due to attenuated insulin-stimulated nitric oxide bioavailability but independent of altered acetylcholine-mediated or sodium nitroprusside-mediated vasodilation. Endothelial cell-specific deletion of Arf6 also resulted in systematic insulin resistance in normal chow-fed mice and glucose intolerance in high-fat diet-fed obese mice. The underlying mechanisms of glucose intolerance were reductions in insulin-stimulated blood flow and glucose uptake in the skeletal muscle and were independent of changes in capillary density or vascular permeability. CONCLUSIONS: Results from this study support the conclusion that endothelial Arf6 signaling is essential for maintaining insulin sensitivity. Reduced expression of endothelial Arf6 impairs insulin-mediated vasodilation and results in systemic insulin resistance. These results have therapeutic implications for diseases that are associated with endothelial cell dysfunction and insulin resistance such as diabetes.

KRAS, MYC, and ARF6: inseparable relationships cooperatively promote cancer malignancy and immune evasion.

Mutations in the KRAS gene and overexpression of protein products of the MYC and ARF6 genes occur frequently in cancer. Here, the inseparable relationships and cooperation of the protein products of these three genes in cancer malignancy and immune evasion are discussed. mRNAs encoded by these genes share the common feature of a G-quadruplex structure, which directs them to be robustly expressed when cellular energy production is increased. These three proteins are also functionally inseparable from each other, as follows.　1) KRAS induces MYC gene expression, and may also promote eIF4A-dependent MYC and ARF6 mRNA translation, 2) MYC induces the expression of genes involved in mitochondrial biogenesis and oxidative phosphorylation, and 3) ARF6 protects mitochondria from oxidative injury. ARF6 may moreover promote cancer invasion and metastasis, and also acidosis and immune checkpoint. Therefore, the inseparable relationships and cooperation of KRAS, MYC, and ARF6 appear to result in the activation of mitochondria and the driving of ARF6-based malignancy and immune evasion. Such adverse associations are frequent in pancreatic cancer, and appear to be further enhanced by TP53 mutations. Video Abstract.

Long non-coding RNA KCNQ1OT1 promotes the progression of gastric cancer via the miR-145-5p/ARF6 axis.

BACKGROUND: Long non-coding RNA KCNQ1 opposite strand/antisense transcript one gene (KCNQ1OT1) has been reported to be involved in the progression of many types of human cancer, whereas its role in gastric cancer (GC) remains unknown. The present study aimed to investigate the role of KCNQ1OT1 in GC. METHODS: In total, 25 GC tissues and adjacent normal tissues were collected. The expression of KCNQ1OT1, miR-145-5p and ARF6 in GC tissues and cell lines was detected by quantitative reverse transcriptase-polymerase chain reaction or western blotting. Bioinformatics analysis and a dual luciferase reporter assay were performed to determine the relationship between KCNQ1OT1 and miR-145-5p or miR-145-5p and ARF6. Gain- and loss-of function of KCNQ1OT1 and miR-145-5p were achieved to confirm their roles in GC cells. Cell counting kit-8, colony formation and flow cytometry assays were used to evaluate cell viability, proliferation and apoptosis. A xenograft tumor model was established with BGC803 tumor cells transfected with sh-KCNQ1OT1 or empty vector to determine the role of LINC01089 in vivo. RESULTS: The expression levels of KCNQ1OT1 were markedly elevated in GC tissues and cells. Knockdown of KCNQ1OT1 inhibited GC tumor growth, reduced GC cell viability and colony formation, and induced GC cell apoptosis. The expression levels of miR-145-5p were significantly decreased in GC cells and correlated with the expression of KCNQ1OT1 in GC tumors. Moreover, KCNQ1OT1 directly binds with miR-145-5p, which is targeting ARF6. Knockdown of KCNQ1OT1 increased the expression levels of miR-145-5p. Inhibition of miR-145-5p increased the expression levels of KCNQ1OT1 and also attenuated the effects of knockdown of KCNQ1OT1 on the viability, proliferation and apoptosis of GC cells. In addition, overexpression of miR-145-5p reduced GC cell viability and colony formation and induced GC cell apoptosis, whereas overexpression of ARF6 attenuated the effects of overexpression of miR-145-5p on GC cell viability, colony formation and apoptosis. CONCLUSIONS: KCNQ1OT1 can promote GC progression through the miR-145-5p/ARF6 axis. KCNQ1OT1 may serve as a therapeutic target and a diagnostic biomarker of GC.

Activation of the GTPase ARF6 regulates invasion of human vascular smooth muscle cells by stimulating MMP14 activity.

Hormones and growth factors stimulate vascular smooth muscle cells (VSMC) invasive capacities during the progression of atherosclerosis. The GTPase ARF6 is an important regulator of migration and proliferation of various cell types, but whether this small G protein can be activated by a variety of stimuli to promote invasion of VSMC remains unknown. Here, we aimed to define whether Platelet-derived growth factor (PDGF), a mitogenic stimulant of vascular tissues, and Angiotensin II (Ang II), a potent vasoactive peptide, can result in the activation of ARF6 in a human model of aortic SMC (HASMC). We demonstrate that these two stimuli can promote loading of GTP on this ARF isoform. Knockdown of ARF6 reduced the ability of both PDGF and Ang II to promote invasion suggesting that this GTPase regulates key molecular mechanisms mediating degradation of the extracellular matrix and migration. We report that PDGF-BB-mediated stimulation of ARF6 results in the activation of the MAPK/ERK1/2, PI3K/AKT and PAK pathways essential for invasion of HASMC. However, Ang II-mediated stimulation of ARF6 only promotes signaling through the MAPK/ERK1/2 and PAK pathways. These ARF6-mediated events lead to activation of MMP14, a membrane-bound collagenase upregulated in atherosclerosis. Moreover, ARF6 depletion decreases the release of MMP2 in the extracellular milieu. Altogether, our findings demonstrate that the GTPase ARF6 acts as a molecular switch to regulate specific signaling pathways that coordinate invasiveness of HASMC.

LncRNA FOXD3-AS1 promotes breast cancer progression by mediating ARF6.

BACKGROUND: Breast cancer is one of the most common malignant tumor in women. The high metastatic characteristics cause a high mortality rate of breast cancer. Increasing number of studies have indicated that long non-coding RNAs (lncRNAs) play key roles in the progression of human cancers including breast cancer. In this study, we studied the expression and molecular mechanisms of lncRNA FOXD3-AS1 in breast cancer. METHODS: The expression of lncRNA FOXD3-AS1 was analyzed by TCGA database and RT-qPCR assay. CCK8 assay was used to measure cell proliferation ability. Cell migration and invasion capacities were detected by transwell assay. Potential targets of lncRNA and miRNA were predicted by bioinformatic tools. The targeting relationship between genes was verified by dual-luciferase reporter assay. The nude mice tumor model was performed to study the effect of FOXD3-AS1 on breast cancer in vivo. Protein expression was detected by western blot. RESULTS: In the present study, we found that the FOXD3-AS1 expression was significantly increased in breast cancer tissues compared with normal tissues and involved in the poor prognosis of patients. Functionally, knockdown of FOXD3-AS1 suppressed cell proliferation and metastasis abilities in vitro, and tumor growth in vivo. Mechanistically, FOXD3-AS1 functioned as a competing endogenous RNA (ceRNA) to upregulate ARF6 expression by targeting miR-127-3p. In addition, the roles of FOXD3-AS1 on cell proliferation and metastasis were achieved through miR-127-3p/ARF6 axis. CONCLUSION: In summary, our results reported the regulatory mechanism of FOXD3-AS1 in breast cancer progression by targeting miR-127-3p/ARF6 axis to affect cell proliferation, migration, invasion and tumor growth.

ADP-ribosylation factor 6 expression increase in oesophageal adenocarcinoma suggests a potential biomarker role for it.

ADP-ribosylation factor 6 small GTPase plays an important role in cell migration, invasion and angiogenesis, which are the hallmarks of cancer. Although alterations in ARF6 expression and activity have been linked to metastatic cancer in one or two tissues, the expression of ARF6 in cancers over a wide range of tissues has not been studied so far. In this report, we analysed the expression of ARF6 mRNA in cancers and corresponding healthy controls from 17 different tissues by real-time qualitative polymerase chain reaction (RT-qPCR). We further evaluated ARF6 protein expression in oesophageal adenocarcinoma (EAC) tissue microarray cores by immunohistochemistry. The ARF6 gene expression levels are highly variable between healthy and cancer tissues. Our findings suggest that the ARF6 gene expression is up-regulated highest in oesophageal cancer. In EAC TMAs, ARF6 protein expression increase correlated with EAC progression. This is the first study to investigate ARF6 gene expression in a wide array of cancer tissues and demonstrate that ARF6 expression, at both mRNA and protein levels, is significantly upregulated in higher grades of EAC, which may be useful in targeting ARF6 for cancer diagnostic and therapeutic purposes.

Rab22a regulates the establishment of epithelial polarity.

Membrane trafficking establishes and maintains epithelial polarity. Rab22a has a polarized distribution in activated T-cells, but its role in epithelial polarity has not been investigated. We showed previously that Rab14 acts upstream of Arf6 to establish the apical membrane initiation site (AMIS), but its interaction with Rab22a is unknown. Here we show that Rab14 and Rab22a colocalize in endosomes of both unpolarized and polarized MDCK cells and Rab22a localizes to the cell:cell interface of polarizing cell pairs. Knockdown of Rab22a results in a multi-lumen phenotype in three-dimensional culture. Further, overexpression of Rab22a in Rab14 knockdown cells rescues the multi-lumen phenotype observed with Rab14 knockdown, suggesting that Rab22a is downstream of Rab14. Because of the relationship between Rab14 and Arf6, we investigated the effect of Rab22a knockdown on Arf6. We find that Rab22a knockdown results in decreased active Arf6 and that Rab22a co-immunoprecipitates with the Arf6 GEF EFA6. In addition, EFA6 is retained in intracellular puncta in Rab22a KD cells. These results suggest that Rab22a acts downstream of Rab14 to traffic EFA6 to the AMIS to regulate Arf6 in the establishment of polarity.

Arf6 exacerbates allergic asthma through cell-to-cell transmission of ASC inflammasomes.

Asthma is a chronic inflammatory disease of the airways associated with excess production of Th2 cytokines and lung eosinophil accumulation. This inflammatory response persists in spite of steroid administration that blocks autocrine/paracrine loops of inflammatory cytokines, and the detailed mechanisms underlying asthma exacerbation remain unclear. Here, we show that asthma exacerbation is triggered by airway macrophages through a prion-like cell-to-cell transmission of extracellular particulates, including ASC protein, that assemble inflammasomes and mediate IL-1ß production. OVA-induced allergic asthma and associated IL-1ß production were alleviated in mice with small GTPase Arf6-deficient macrophages. The extracellular ASC specks were slightly engulfed by Arf6-/- macrophages, and the IL-1ß production was reduced in Arf6-/- macrophages compared with that in WT macrophages. Furthermore, pharmacological inhibition of the Arf6 guanine nucleotide exchange factor suppressed asthma-like allergic inflammation in OVA-challenged WT mice. Collectively, the Arf6-dependent intercellular transmission of extracellular ASC specks contributes to the amplification of allergic inflammation and subsequent asthma exacerbation.